The main aim of this work was to investigate the effect of [NCO]/[OH] molar ratio used during the prepolymer chain extending step (with bio‐based diols) on the chemical structure, and thermomechanical and mechanical properties of thermoplastic poly(ether‐urethane)s. Thermoplastic poly(ether‐urethane)s were obtained from bio‐based polyol (polytrimethylene ether glycol), bio‐based glycol (1,4‐butanediol or 1,3‐propanediol), and 4,4'diphenylmethane diisocyanate via a prepolymer route. In the first step, the prepolymer was synthesized from a polyol and diisocyanate. In the second step, the prepared prepolymer terminated with isocyanate group had been extended by using bio‐glycol at three different [NCO]/[OH] molar ratios, namely, 0.9, 0.95, and 1.0. The chemical structure was analyzed using Fourier transform infrared spectroscopy. The Gaussian deconvolution technique was used to study the hydrogen bonding as well as to decompose carbonyl region of three peaks in various TPUs. It has been confirmed that the glycol type and [NCO]/[OH] molar ratio have an effect on the degree of phase separation, and thermomechanical and mechanical properties of resulting materials. It was also observed that the degree of microphase separation increased with increasing content of 1,3‐propanediol used as a chain extender. Materials with the highest [NCO]/[OH] ratio showed the most favorable mechanical properties. POLYM. ENG. SCI., 58:E199–E206, 2018. © 2018 Society of Plastics Engineers